The Impact of Genetic Background and Cell Lineage on the Level and Pattern of Position Effect Variegation

Background Chromatin-based transcriptional silencing is often described as a stochastic process, largely because of the mosaic expression observed in position effect variegation (PEV), where a euchromatic reporter gene is juxtaposed with heterochromatin. Here we closely examine the impact of genetic background on PEV phenotypes in the fruit fly, Drosophila melanogaster. Results Using consecutive generations of selective breeding, we isolated, from a single laboratory population, two inbred lines exhibiting contrasting degrees of variegation (A1: low expression, D1: high expression). Within each inbred population, remarkable similarity is observed in both the degree and the pattern of variegation. 89.63% of the differences between the two inbred lines in the degree of silencing can be explained by genotype, while a modest but significant sex effect is also observed. Further analyses of the PEV phenotype in the progeny of crosses between A1 and D1 suggest that the genotypic effect is the result of the combined effect of multiple independent trans-acting loci. While the initial observations are based on a PEV phenotype scored in the fly eye (hsp70-white reporter), similar degrees of silencing were observed using a beta-gal reporter that can be scored across the whole fly. The pattern of variegating hsp70-white expression among individual flies becomes almost identical after five generations of inbreeding. Using a reporter inserted into the heterochromatic fourth chromosome, image analysis found significant enrichment of pigmentation in the ventral-posterior quadrant in both the A1 and D1 lines, and in the F1 and F2 progeny produced from a cross between A1 and D1, despite different degrees of expression. Conclusions Combining these results with the spreading model for local heterochromatin formation, we propose an augmented stochastic model to describe PEV. In this model, the genetic background, which determines the overall level of silencing, works with the cell lineage specific regulatory environment to determine the on/ off probability at the reporter locus in each cell. This model acknowledges cell-type specific events, as well as the general impact of heterochromatin formation.

quadrant in both the A1 and D1 lines, and in the F1 and F2 progeny produced from a cross between A1 and D1, despite different degrees of expression.

Conclusions:
Combining these results with the spreading model for local heterochromatin formation, we propose an augmented stochastic model to describe PEV. In this model, the genetic background, which determines the overall level of silencing, works with the cell lineage specific regulatory environment to determine the on/ off probability at the reporter locus in each cell. This model acknowledges cell-type specific events, as well as the general impact of heterochromatin formation.

Background
Position effect variegation (PEV) describes the mosaic expression of a phenotype in a cell population that is otherwise thought to be uniform. It has generally been studied in cases where the cell-autonomous phenotype is easily visualized, such as eye pigmentation, but appears to be a general phenomenon [1,2]. Muller reported the original observation of variegating eye pigmentation in adult flies, recovered following X-ray mutagenesis. Because of the high degree of variation in the pattern, and in the level of pigmentation between individuals and across generations, he described the phenotype as "ever sporting" [3]. The report on this highly variable phenotype led to various speculative models describing how such a heritable, yet variable phenotype could arise [4]. Further investigations have led to a generally accepted transcriptional silencing model based on a stochastic spreading of heterochromatin [5]. The X-rayinduced inversion generated by Muller juxtaposed the white gene, which is required cellautonomously for proper deposition of eye pigment, with the pericentric heterochromatin. The spreading of pericentric heterochromatin packaging to the white locus results in concomitant silencing; when this occurs in some but not all of the cells, the result is a variegated pattern of eye pigmentation. This spreading process is thought to be stochastic (reviewed in [6]).
Screens for second-site modifiers of a PEV phenotype have identified numerous loci that have a strong impact on the expression level of the PEV phenotype for the cell population as a whole. These genetic modifiers are referred to as suppressors [Su(var); loss of silencing]] or enhancers [E(var); gain in silencing] of PEV (see [2,6] for review).
Some of these loci exhibit antipodal effects, i.e. if one dose of the gene results in loss of silencing, three doses results in an increase in silencing. This antipodal response has been interpreted as evidence that the probability of the heterochromatin spreading process occurring is determined at least in part by the dosage of key gene products that constitute the structural components of heterochromatin; in other cases an enzymatic contribution is implied. An assay for a PEV phenotype following a one generation cross to assess dominant effects of candidate PEV modifier alleles has therefore been commonly used to test the participation of a given gene of interest in the process of heterochromatin formation and gene silencing [7]. In fact, screens for PEV suppressors have been a major source of candidate genes for further analysis of the process of heterochromatin formation [8,9].
We previously devised a P element reporter to probe the heterochromatin landscape of the genome, P{hsp26-pt, hsp70-white}. Using the well-characterized hsp70 promoter to drive a white reporter gene, about 1 % of the insertion lines recovered following mobilization exhibit a variegating eye phenotype [10]. Mapping of these variegating insertion lines revealed an outline of heterochromatin distribution in the genome, which is in agreement with prior cytological assignments, but provides higher resolution. PEV is observed following insertion of the reporter P element into the pericentric and telomeric regions of the major autosomes and the X chromosome, as well as regions of the Y chromosome. Based on the eye phenotype, the fourth chromosome (Muller F element), while largely heterochromatic, appears to have interspersed heterochromatic and permissive domains [11]. Characterization of these variegating P element reporter insertion lines has indicated that the basic principles for variegation as observed in the original white mottled line from Muller (i.e. sensitivity to sex chromosome dosage, etc.) are common to most variegating lines [10], although individual heterochromatic domains can show differences in sensitivity to a subset of the known suppressors of variegation [12][13][14]. A major exception are insertions into the TAS (Telomere Associated Sequences) sequences, just proximal to HeT-A and TART; these lines exhibit a PEV phenotype that is sensitive to mutations in the Polycomb silencing machinery [15], while ChIP analysis shows association with Pc [16]. Here we have used as our test locus a reporter in the fourth chromosome, a largely heterochromatic domain that for the most part mimics pericentric heterochromatin, a chromatin structure dependent on H3K9 methylation and HP1a [10,12].
Although PEV has been tremendously helpful in developing our understanding of heterochromatin, its stochastic nature continues to raise unanswered questions.
Numerous mutations have been identified to modify PEV; it is estimated that there are more than 150 such modifiers in the fly genome [17]. Genetic background -including different assortment of alleles at these many loci -could affect the probability for a spreading event to occur in a given fly within a stock, and thus could contribute to the variation seen in PEV phenotypes. In fact, a recent study looking at PEV in an outbred fly population suggested that many more modifier loci likely exist across the genome [18]. Here, we examine the variation in PEV phenotype in a single laboratory reporter line using P{hsp26-pt, hsp70-white}. The study was carried out using a 4 th chromosome PEV reporter line, 39C-12, for several reasons. 39C-12 is relatively well-characterized in terms of its response to PEV modifiers [12,13,19]. Its position has been mapped to a precise location in the genome [11]. The hsp70 promoter used in this reporter is wellcharacterized [20]. It's basal activity at 25°C in this construct is sufficient to cause a uniform red eye when the reporter is inserted into a euchromatic site. Finally, the 39C-12 stock is considered relatively "clean," because of the genetic bottleneck that occurred during the production of the transgenic line; specifically, the line is derived from a single male with the P element insertion on the fourth chromosome, backcrossed to y w 67c23 females. The starting stock of 39C-12 had a high level of variation in PEV. We found that much of the variation in the strength of the PEV phenotype (i.e. the degree of silencing) for this stock could be attributed to genetic variants floating in the background. Intriguingly, we found fairly consistent patterns in PEV phenotype among individual flies across the inbred populations, indicating a more controlled expression of the PEV phenotype between cells than a purely stochastic heterochromatin spreading model would otherwise suggest. Our observations resonate well with published literature and provide fresh insights into this classic system. population, in opposite directions). In addition to the genetic effect introduced by selective breeding, there is also a sex effect impacting the PEV phenotype. While there is a modest 26.02% higher pigment level in A1 males (relative to A1 females, p < 0.05), there is a 47.93% higher pigment level in D1 males compare to D1 females (p < 0.001).

Visual inspection shows considerable variation in
Overall, the combined genotype and sex effects explain 95.9% of the variance between A1 and D1 flies, while genotype alone explains 89.6% of the variance.
To assess the genetic architecture of the two inbred lines regarding its impact on the PEV phenotype, crosses were performed between these lines to generate F1 and The results above are based on the PEV eye phenotype of a P-element hsp70white reporter inserted into the heterochromatic 4 th chromosome. To determine whether the conclusions drawn are generally applicable to the PEV phenotype, we evaluated the impact of the A1 and D1 background genotypes on the PEV phenotype of a Y-linked hsp70-LacZ PEV reporter, Tp(3;Y)BL2 (BL2). The PEV phenotype of the BL2 LacZ reporter line used for this purpose results from a translocation of a fragment of the 3 rd chromosome carrying the reporter to the Y chromosome following X-ray irradiation [21].
A multigeneration cross scheme was designed to introduce this Y-linked PEV reporter into the A1 (or D1) genotype, without perturbing that genetic background, utilizing dominantly marked balancer chromosomes (and the fact that meiotic recombination is not known to occur in the male germ line [22,23]) (Supp. Fig 1). Two inbred lines containing the Y-linked BL2 reporter in the A1 and D1 genetic backgrounds, respectively, were derived (BL2-A1 and BL2-D1). The level of beta-galactosidase activity (mAU/min) in lysates prepared from single male flies was used as a quantitative readout for the PEV phenotype. A consistent PEV phenotype for the BL2 reporter across individuals from each of the A1 or D1 genetic backgrounds was observed, with D1 flies exhibiting ~4.64 times the activity of A1 flies (Fig 3). The BL2 reporter in a D1  Table 2 for a summary) suggests differences in the ability of cells in the ventral-posterior quadrant and cells in the rest of the eye to silence the reporter. These results argue that nonrandom variation in heterochromatin silencing among cells plays a role in determining the observed pattern.

Discussion
A high level of heterogeneity in PEV phenotypes is commonly observed within laboratory reporter strains, even when the genetic background is standardized (as was the case here) by back crossing to yw 67c23 . Through selective breeding, we successfully derived inbred fly lines from the 39C-12 parent stock that show consistently strong or weak PEV (lines A1 and D1). Further analysis of the PEV phenotype from a twogeneration cross between the two inbred lines indicates that there are likely multiple trans genetic modifier loci in the background acting independently to determine the strength of PEV. This conclusion is reinforced by results from analyzing the Y-linked BL2 reporter; there, a similar impact of the A1 or D1 genotype was observed on a different reporter, which removes the possibility of cis-linkage between the reporter and the background PEV modifier variants. These results resonate well with a recent study on PEV phenotype using an outbred population [18]. There, Kelsey and Clark found a large number of genetic variants across the genome significantly associated with the strength of PEV. Here, we have demonstrated that even within a relatively inbred population, one still observes a high level of variation in phenotype, which is best described by the effects from multiple independent modifier loci.
While this study mostly focused on the genetic background of a single reporter line, 39C-12, this level of heterogeneity in PEV phenotype is commonly observed in our laboratory across multiple reporter lines. Given that we successfully derived two inbred lines that have more than 3-fold difference in eye pigment level from a single parent population, it is worthwhile considering the implications for using a reverse genetic candidate approach for identifying novel PEV modifiers [2,6]. In addition to forward genetic screens, novel PEV modifiers are often identified through a reverse genetic screen for dominant effects on PEV using mutant alleles of genes that are suspected to play a role in chromatin-based transcriptional silencing (e.g. SETDB1, G9a, PIWI). High levels of variation in the starting reporter PEV line often result in high levels of variation in the readout for the screen; some researchers will therefore decide to homogenize the genetic background of the starting reporter line to reduce variation in order to gain power to detect dominant effects from the mutant alleles being tested. In light of the results presented here, the steps researchers take to homogenize the background could lead them to unwittingly enrich or deplete the genetic background of variants that might interact with the mutant allele of interest, and therefore amplify or reduce the phenotypic impact. This practice could therefore lead to inconsistent findings between labs and potentially misleading results. For more reproducible findings, instead of homogenizing the genetic background, researchers could increase power by increasing sample size. This suggestion is particularly appropriate for model organism researchers, such as fly geneticists, considering the limited cost imposed by increasing sample size for a pigment assay or an enzymatic assay for assessing PEV phenotype, even by 5 or 10 times the current standard.
The results using the BL2 reporter on the Y chromosome reproduced the results  [10,26]. This study followed up on observations of a PEV pattern in the 39C-12 reporter line through selective breeding and then formally tested the enrichment pattern through statistical analysis on images of the PEV phenotype. The results suggest a more controlled reporter expression than would be inferred from a random spreading of heterochromatin. While the random spreading model is adequate for explaining sectors of PEV reporter expression in S. pombe colonies [1,5], in higher eukaryotes, it appears that a more elaborate model considering developmental lineage differences between cells displaying the phenotype is needed to adequately describe the process.
Consistent with this conjecture, we did not find the Y chromosome BL2 reporter, which is in a different cis-regulatory environment and likely subject to different regulation during the process of developmental lineage differentiation, to express similar patterns of PEV phenotype as the 39C-12 reporter, despite having been transferred into the same A1 or D1 genetic background. One suggestion as to the source of differences in PEV patterns between cell types is the timing of the last wave of cell divisions during metamorphosis. Using the beta-gal reporter, Lu et al. reported that in the eye disk, there is a dramatic difference in variegation on either side of the morphogenetic furrow, with a relaxation of silencing at this juncture [24]. Such features could be a contributor, as cells that go through an S phase later in developmental time are inherently exposed to a different environment during this process.

Conclusions
In summary, our observations with two PEV reporters inserted in different cis-regulatory environments in two diverging trans-acting genetic backgrounds indicate that while trans-acting modifiers play the major role in determining the degree of PEV silencing, the pattern of PEV silencing is likely influenced more by the cis-regulatory environment of the reporter insertion site.

Fly husbandry and genetics
Flies were cultured at 25°C, 70% humidity on regular cornmeal sucrose-based medium [27]. Unless otherwise specified, genetic crosses were performed by mating 2 male flies to 3~5 female virgin flies. The 39C-12 reporter line [10] was used as the starting line to generate A1 and D1 inbred lines. Five generations of consecutive full sibling crosses with selection for extreme eye phenotype at each generation were performed to create the two inbred lines. To substitute the BL2 Y-linked PEV reporter [21] into the A1 or D1 genetic background, dominantly marked balancers were used to follow the second and third chromosomes (see Supp . Fig 1 for crossing scheme). Balancers SM5 and TM6 were first introduced to the BL2 line by a standard cross, and the F1 male progeny that had both second and third chromosomes balanced were selected (based on the dominant markers) to mate with female flies from the inbred line. A male F2 progeny from the F1 cross with both balancers over inbred chromosomes were selected to backcross to 3~5 inbred line female virgins. Only one male fly was used in the F2 cross in order to ensure that there would only be two genotypes of 4 th chromosome in the F3 population (i.e. the original 39C-12 4 th chromosome and the other 4 th chromosome, which is denoted as + iso in Supp. Fig 1, carried by the selected F2 male). Because the 4 th chromosome is not known to recombine during meiosis (or does so extremely rarely), progeny from the backcross lacking both balancers were selected to make a floating stock (i.e. inbred genetic background with an unmarked 4 th chromosome floating). This floating stock was made homozygous for the 39C-12 4 th chromosome, as judged by the presence of the 39C-12 reporter expression, to generate the final stock.
In order to evaluate the homozygosity of the 4 th chromosome in the final stock, 39C-12 reporter expression in all female progeny was followed by visual inspection for multiple generations. (white expression in male progeny cannot be used to evaluate 39C-12 expression because of the interference coming from the mini-white construct in the BL2 reporter.)

PEV assays
Eye pigment extraction and quantification was done essentially as previously described [28] with a few modifications. Instead of hand homogenizing for pigment extraction, whole flies were homogenized using a Mixer Mill Mm 300 to increase the throughput and consistency. The overnight incubation at 4°C was omitted. For each genotype of each sex, 20~30 age-matched flies (3~5-day-old) were randomly selected from the population and sorted according to their pigmentation level by visual inspection. Five flies of similar pigmentation levels were than collected together as one sample. The same protocol was used for both sex.
X-gal staining of eye imaginal discs and the assay of beta-galactosidase activity were carried out as previously described [29].
For image analysis of the PEV phenotype, eye pictures from a random sampling of the PEV phenotype from each population were selected based on eye size and angle of the photo to ensure consistent quantification. Each image was then converted to 8-bit grey scale in imageJ, and then further converted to a binary image through manual threshold setting. The guideline used for threshold setting was to capture as many variegating speckles as possible without introducing large patches of shadow that resulted from lighting differences during imaging. The area to quantify was manually selected using the imageJ oval tool to cover as much of the eye as possible without covering other anatomical structures. The binary oval image representation of the eye PEV phenotype was then converted into a binary table of quantification using the imageJ image to result function, where each entry in the table represents a pixel in the image. To test for enrichment of pigmentation in the ventral-posterior quadrant, the proportion of "expressed" pixels in the ventral-posterior quadrant was compared to the proportion of "expressed" pixels outside of the quadrant of interest using statistical tests described in the following section.

Statistical analysis
Analysis of the strength of the PEV phenotype (e.g. pigment level) was done using either R or excel. For estimating the genotype effect and the sex effect on the variation in PEV phenotype between the A1 and D1 lines, we fit the pigment assay data to a linear model using the genotype label and sex label as predictors. More precisely, the OD 480 reading for pigment level was first log transformed and then fitted as the response variable in the following linear model using the lm() function in R: where P i represents the pigment level of individual i, G i is the indicator variable for the genotype label, and S i is the indicator variable for the sex label. The adjusted R^2 produced by applying the summary() function to the above described lm object is used to estimate the percentage variance explained by the model. To evaluate the significance of the genotype effect (i.e. the differences in PEV phenotype between the A1 and D1 genotypes) we performed an F test by applying the anova() function on the lm object. Analysis of the pattern of PEV followed a similar linear model framework, where the proportion of pigmentation in or out of the ventral-posterior quadrant is modeled using a binary predictor of location. To evaluate the association between the proportion of pigmentation and the location we performed an F test.

Declarations
Ethics approval and consent to participate Not applicable

Consent for publication
Not applicable Availability of data and material The data used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
The funding support for this study came from a Howard A. Schneiderman Fellowship to SHW and an NIH grant GM068388 to SCRE. The funding agencies exerted no influence in the design of the study, data collection, data analysis, the interpretation of results, and the preparation of the manuscript.
Authors' contributions SHW conceived the idea for this study. SHW and SCRE designed the study. SHW executed the experiments and data collection. SHW analyzed the data. SHW and SCRE interpreted the results and wrote the manuscript.      image was first rotated so that the maximal area of the fly eye could be selected using the oval tool. Pixels outside the selected oval area were removed (pseudo-colored in grey for illustration) while pixels within the oval area were converted to binary by setting a threshold. The threshold selection was done manually to best represent the original eye phenotype. To evaluate the similarity of the PEV pattern between individuals, each image of a fly eye was split into four even quadrants (blue lines) and the pigment enrichment (i.e. the proportion of black pixels) in the ventral-posterior quadrant (red arrow) was evaluated against the area outside the ventral-posterior quadrant. Tables   Table 1. Pigment assay results for 39C-12 inbred variegating lines * Average values reported for measurements of pigment level; each measurement was obtained from pigment extracted from a pool of five representative flies as one sample.   BL2/yw A1 ; + A1 ; + A1 ; 39C-12/+ iso X yw A1 ; + A1 ; + A1 ; 39C-12/+ iso BL2-A1 stock with + iso floating Supp. Figure 1 A B C